• Tryptone (Oxoid, cat. no. LP0042)
• Yeast extract (Oxoid, cat. no. LP0021)
• Bis-Tris (Sangon Biotech, cat. no. A610079-0100)
• EDTA (Sangon Biotech, cat. no. A100105-0500)
• Thioflavin T (Sigma Aldrich, cat. no. T3516)
• Proteinase K (Invitrogen, cat. no. 25530031)
• NaN3 (Sigma Aldrich, cat. no. S2002)
• NaCl (Sangon Biotech, cat. no. A100241-0500)
• KCl (Sangon Biotech, cat. no. A501159-0500)
• HCl (Hushi, cat. no. 10011018)
[CAUTION!] HCl is highly toxic, corrosive, unstable and explosive. Avoid flames and handle in a fume hood. Be sure to wear lab-gowns, masks and gloves when operating. Dispose should be in accordance with local regulations.
• PBS (Sangon Biotech, cat. no. E607008-0500)
• Tris (Sangon Biotech, cat. no. A501492-0500)
• IPTG (Sangon Biotech, cat. no. A600168-0100)
• SDS (Sangon Biotech, cat. no. A600485-0500)
• Glycerol (Hushi, cat. no. 10010618)
• Streptomycin sulfate (A610494-0250)
• PMSF (Sangon Biotech, cat. no. A610425-0025)
[CAUTION!] PMSF is toxic. Handle with protective gloves.
• Isopropanol (Hushi, cat. no. 80109218)
• β-Mercaptoethanol (Sigma Aldrich, cat. no. M6250-500ML)
[CAUTION!] β-Mercaptoethanol is volatile with a pungent odor. Make sure that wear a mask and operate in the fume hood during experiments. Dispose should be in accordance with local regulations.
• Copper grids (200 mesh) with coated carbon support film (Beijing Zhongjingkeyi Technology, cat. no. BZ110205b)
• 0.22-μm centrifugal filter (Millipore, cat. no. SLGP033NS)
• Bis-Tris gel (GenScript, cat. no. M00656)
• Q column (GE Healthcare, cat. no. 17-5156-01)
• Superdex 75 (GE Healthcare, cat. no. 28-9893-33)
• 96-well plate (Corning, cat. no. 3590)
• 384-well optical plate (Thermo Scientific, cat. no. 142761)
• Dialysis Clip (Thermo Scientific, cat. no. 87733)
• Filter paper (Jiaojie, GB/T1914-2007)
• Centrifugal filters 3K (Millipore, cat. no. UFC900396)
• 1.5-mL microtube (Axygen, cat. no. MCT-150-C)
• 15-mL Centrifuge tube (YUEYI, cat. no. YB0019-15)
• 50-mL Centrifuge tube (YUEYI, cat. no. YB-50D)
• 2× YT medium. Dissolve 16 g Tryptone, 10 g Yeast extract and 5 g NaCl with deionized water. Bring the volume to 1 L and perform high temperature sterilization. This medium can be stored at room temperature (RT).
• 1 mol/L Tris (pH 8.0) stock. Dissolve 121.14 g Tris in 900 mL deionized water and stir until most of it is dissolved. Adjust the pH to 8.0 with HCl. Bring the volume to 1 L and filter-sterilize the solution. This solution can be stored at RT.
• 3 mol/L KCl stock. Dissolve 223.65 g KCl in 900 mL deionized water and stir until most of it is dissolved. Bring the volume to 1 L and filter-sterilize the solution. This solution can be stored at RT.
• 4 mol/L NaCl stock. Dissolve 233.76 g NaCl in 900 mL deionized water and stir until most of it is dissolved. Bring the volume to 1 L and filter-sterilize the solution. This solution can be stored at RT.
• 1 mol/L Tris (pH 7.5) stock. Dissolve 121.14 g Tris in 900 mL deionized water and stir until most of it is dissolved. Adjust the pH to 7.5 with HCl. Bring the volume to 1 L and filter-sterilize the solution. This solution can be stored at RT.
• 500 mmol/LM EDTA Stock. Dissolve 186.12 g EDTA in 900 mL deionized water and stir until most of it is dissolved. Bring the volume to 1 L and filter-sterilize the solution. This solution can be stored at RT.
• Bacteria lysis buffer. 100 mmol/L Tris (pH 8.0), 1 mmol/L EDTA,1 mmol/L PMSF.
• Bacteria storage buffer. 100 mmol/L Tris (pH 8.0), 1 mmol/L EDTA.
• Buffer A. 25 mmol/L Tris (pH 8.0). This solution can be stored at RT.
[CAUTION!] Before each use, ultrasonic debubble and filtration are a must.
• Buffer B. 25 mmol/L Tris (pH 8.0), 1 mol/L NaCl. This solution can be stored at RT.
[CAUTION!] Before each use, ultrasonic debubble and filtration are a must.
• Buffer for size exclusion chromatography. 50 mmol/L Tris (pH 7.5), 150 mmol/L KCl.
[CAUTION!] Ultrasonic debubble and filtration must be performed before each use.
• Fibril growth and ThT buffer. 50 mmol/L Tris (pH 7.5), 150 mmol/L KCl, 0.05% NaN3.
• Loading buffer (5×). Dissolve 5.0 g SDS in 8.33 mL 1.5 mol/L Tris (pH 6.8). Then add 25 mL glycerol, avoiding air bubbles, and bring the volumn to 45 mL with deionized water. After complete dissolution, add 250 mg bromophenol blue and bring the volumn to 50 mL with β-mercaptoethanol.
[CAUTION!] β-Mercaptoethanol is volatile with a pungent odor. Make sure that wear a mask and operate in the fume hood during experiments. Dispose should be in accordance with local regulations.
• PMSF (0.2 mol/L). Dissolve 0.70 g PMSF in 16 mL isopropanol. After ultrasonic dissolution, bring the volumn to 20 mL. 1 mL aliquots should be protected from light and stored at –20 °C. PMSF can be stored for one year.
[CAUTION!] PMSF is acutely toxic. Wear a mask and handle with protective gloves.
• Insoluble buffer. 50 mmol/L Tris, pH 8.0, 150 mmol/L NaCl, 1% Triton X-100, 2% SDS.
[CAUTION!] SDS is harmful. Handle with protective gloves.
• IPTG (1mol/L). Dissolve 23.83 g IPTG in 90 mL deionized water. After complete dissolution, bring the volumn to 100 mL. Aliquots should be stored at –20 °C.
• SDS-PAGE running buffer (1×). Add 25 mmol/L Tris, 192 mmol/L glycine and 0.1% (w/v) SDS; bring it to the final volume with water. This buffer can be stored indefinitely at RT.
• ThT solution (1 mmol/L). Dissolve 3.19 mg ThT in 9 mL deionized water. After completely dissolved, bring the volumn to 10 mL. Aliquots should be stored at –4 °C.